PROJECT SUMMARY/ABSTRACT The overall objectives of this R03 proposal are to investigate the pro-osteogenic effects of NGF in dental pulp stem cells (DPSCs) in vitro and in vivo and to apply this knowledge to foster the application of DPSCs in multiple clinical settings. DPSCs are multipotent progenitor cells that are readily available from extracted or exfoliated teeth and possess high mineralization potential and proliferation rates. As such, DPSC-mediated therapy has produced clinical success in regenerating bone defects and consequently holds great promise for craniofacial disorders. Our recent work in isolating homogenous populations of DPSCs using cell surface marker expression revealed that CD271 was the most reliable marker to isolate DPSCs with high osteogenic potential. Furthermore, treatment with NGF, a known ligand for CD271, promoted osteogenic differentiation of DPSCs. When we examined the intracellular signaling pathways of NGF, we found that ALK and JNK were upregulated during NGF-mediated DPSC osteogenesis, confirming receptor based signal transduction. Upon examining epigenetic regulators, we found that NGF significantly induced expression of KDM4B, a histone demethylase responsible for removing the silencing mark, H3K9me3. We therefore hypothesize that NGF enhances osteogenic differentiation of DPSCs through CD271 signaling and epigenetic modulation of osteogenic genes (e.g., RUNX2 and DLX5). This hypothesis will be tested by the following specific aims: 1) To determine the functional involvement of CD271 receptor and delineate its intracellular signaling in NGF- induced osteogenic differentiation in DPSCs both in vitro and in vivo; 2) To elucidate the role of epigenetic regulators in NGF-mediated DPSC differentiation. The current proposal holds the promise of revealing the mechanistic insights of NGF-mediated osteogenesis in DPSCs, with significant implications for craniofacial tissue engineering. Combined DPSC and NGF therapy may be the innovative answer for the future of regenerative therapy on the basis of not only its superior osteogenic capacity but also its role in neurogenesis and angiogenesis, all of which create a favorable microenvironment for regeneration. Summary: The current R03 proposal is formulated to accumulate preliminary data for subsequent R01 application. Future studies in R01 will combine two emerging areas, epigenetics and tissue engineering using dental MSCs, in the development of clinical tools in craniofacial defect regeneration. Additional roles of epigenetics in dental MSC function and differentiation will be explored, elucidating the complex interplay between various epigenetic modulators and the synergistic interactions between epigenetic and genetic factors. KDM4B knockout mice will be evaluated to determine the role of KDM4B in craniofacial development. Additionally, using the mouse model from this R03 proposal, KDM4B's role in bone tissue engineering will also be assessed.